GSM7384003: T cells, NY-ESO-1 TCR + library knockin, input, donor 2; Homo sapiens; RNA-Seq
Chromium Single Cell 5' Reagent Kit, v1 chemistry (10x Genomics) was used according to the manufacturer's protocol. After GEM (Gel Bead-In Emulsions) recovery, the mRNA library was converted to cDNA, amplified for 11 cycles, and quantified with Agilent Bioanalyzer High Sensitivity. 75% of the amplified cDNA material was carried through for transcriptome library preparation according to the manufacturer's protocol. The remaining 25% of amplified cDNA was used for amplicon sequencing of the knockin barcodes. The cDNA was enriched for knockin barcodes using a nested PCR strategy with Kapa HiFi HotStart Ready Mix (Roche) for 8 cycles per round. For the first PCR, 0.5µM each of ModPoKI_Seq_1_forw primer and ModPoKI_Seq_1_rev primer was used. Amplified products were purified with 0.8x SPRIselect Reagent Kit (Beckman Coulter) and eluted in 10µL nuclease-free water. The libraries were further enriched with a second PCR using 0.5µM each of ModPoKI_Seq_2_forw primer and ModPoKI_Seq_2_rev primer. Amplified products were purified with 0.8x SPRIselect Reagent Kit (Beckman Coulter) and eluted in 15µL nuclease-free water. Lastly, index PCR was performed with Kapa HiFi HotStart Ready Mix (Roche) for 8 cycles with 2.5µL each Nextera Chromium i7 Sample Indices N Set A (PN 3000262) and 0.5µM ModPoKI_Seq_index primer. Amplified products were purified with 0.8x SPRIselect Reagent Kit (Beckman Coulter) and eluted in 45µL nuclease-free water. Samples were pooled and sequenced on a NovaSeq S4 flow cell with 20% PhiX using read parameters 30x8x98. ModPoKI-Seq